Name: measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo
Uploaded: 2014-03-29T14:00:00
Description: Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

We thank Manfred Heckmann for helpful discussions during establishing of the method. Further, we thank Claudia Sommer for technical assistance and Frank Schubert for support producing the video. The work was supported by the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1002 and the Interdisciplinary Center for Clinical Research (IZKF) of Jena University Hospital.

All experimental procedures mentioned in the following protocol were approved by the Thuringian state authorities (Th&#252;ringer Landesamt f&#252;r Verbraucherschutz, Reg.-Nr. 02-044/12).
1. Preparations for Experiment
<ol>
	<li>Fabrication of suction electrodes
	<ol>
		<li>Pull a micropipette using a standard borosilicate glass capillary with a micropipette puller, e.g. a standard patch electrode.</li>
		<li>Brake the electrode to tip diameter of 0.5-1 mm (slightly larger than the...

<ol>
	<li>Eccles, J. C., Eccles, R. M., Magni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+inhibitory+action+attributable+to+presynaptic+depolarization+produced+by+muscle+afferent+volleys.">Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys.</a> J. Physiol. 159, 147-166 (1961).</li><li>Levy, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+gaba+in+primary+afferent+depolarization.">The role of gaba in primary afferent depolarization.</a> Prog. Neurobiol. 9, 211-267 (1977).</li><li>Eccles, J. C., Magni, F., Willis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization+of+central+terminals+of+Group+I+afferent+fibres+from+muscle.">Depolarization of central terminals of Group I afferent fibres from muscle.</a> J. Physiol. 160, 62-93 (1962).</li><li>Eccles, J. C., Schmidt, R., Willis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Studies+on+Presynaptic+Inhibition.">Pharmacological Studies on Presynaptic Inhibition.</a> J. Physiol. 168, 500-530 (1963).</li><li>Maxwell, D. J., Bannatyne, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+muscle+spindle+afferent+terminations+in+lamina+VI+of+the+cat+spinal+cord.">Ultrastructure of muscle spindle afferent terminations in lamina VI of the cat spinal cord.</a> Brain Res. 288, 297-301 (1983).</li><li>Barber, R. P., Vaughn, J. E., Saito, K., McLaughlin, B. J., Roberts, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+terminals+are+presynaptic+to+primary+afferent+terminals+in+the+substantia+gelatinosa+of+the+rat+spinal+cord.">GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord.</a> Brain Res. 141, 35-55 (1978).</li><li>Wall, P. 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Biol. 87, 33-46 (2005).</li><li>Hochman, S., Shreckengost, J., Kimura, H., Quevedo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+inhibition+of+primary+afferents+by+depolarization:+observations+supporting+nontraditional+mechanisms.">Presynaptic inhibition of primary afferents by depolarization: observations supporting nontraditional mechanisms.</a> Ann. N.Y. Acad. Sci. 1198, 140-152 (2010).</li><li>Thompson, S. W., Wall, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+GABA+and+5-HT+receptor+antagonists+on+rat+dorsal+root+potentials.">The effect of GABA and 5-HT receptor antagonists on rat dorsal root potentials.</a> Neurosci. Lett. 217, 153-156 (1996).</li><li>Enriquez-Denton, M., Manjarrez, E., Rudomin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+PAD+and+presynaptic+inhibition+of+muscle+spindle+afferents+after+peripheral+nerve+crush.">Persistence of PAD and presynaptic inhibition of muscle spindle afferents after peripheral nerve crush.</a> Brain Res. 1027, 179-187 (2004).</li><li>Wall, P. D., Devor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+peripheral+nerve+injury+on+dorsal+root+potentials+and+on+transmission+of+afferent+signals+into+the+spinal+cord.">The effect of peripheral nerve injury on dorsal root potentials and on transmission of afferent signals into the spinal cord.</a> Brain Res. 209, 95-111 (1981).</li><li>Witschi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+&#945;2-GABAA+Receptors+in+Primary+Afferent+Depolarization+and+Spinal+Pain+Control.">Presynaptic &#945;2-GABAA Receptors in Primary Afferent Depolarization and Spinal Pain Control.</a> J. Neurosci. 31, 8134-8142 (2011).</li><li>Calancie, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+alterations+in+presynaptic+inhibition+contribute+to+segmental+hypo-+and+hyperexcitability+after+spinal+cord+injury+in.">Evidence that alterations in presynaptic inhibition contribute to segmental hypo- and hyperexcitability after spinal cord injury in.</a> 89, 177-186 (1993).</li><li>Geis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiff+person+syndrome-associated+autoantibodies+to+amphiphysin+mediate+reduced+GABAergic+inhibition.">Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition.</a> Brain. 133, 3166-3180 (2010).</li><li>Geis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+IgG+directed+against+amphiphysin+induces+anxiety+behavior+in+a+rat+model+after+intrathecal+passive+transfer.">Human IgG directed against amphiphysin induces anxiety behavior in a rat model after intrathecal passive transfer.</a> J. Neural Transm. 119 (8), 981-985 (2012).</li><li>Barron, D. H., Matthews, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interpretation+of+potential+changes+in+the+spinal+cord.">The interpretation of potential changes in the spinal cord.</a> J. Physiol. 92, 276-321 (1938).</li><li>Schmidt, R. F., Trautwein, W., Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+root+potentials+evoked+by+natural+stimulation+of+cutaneous+afferents.">Dorsal root potentials evoked by natural stimulation of cutaneous afferents.</a> Nature. 212, 522-523 (1966).</li><li>Eccles, J. C., Schmidt, R. F., Willis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+inhibition+of+the+spinal+monosynaptic+reflex+pathway.">Presynaptic inhibition of the spinal monosynaptic reflex pathway.</a> J. Physiol. 161, 282-297 (1962).</li><li>Manjarrez, E., Rojas-Piloni, J. G., Jimenez, I., Rudomin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+synaptic+transmission+from+segmental+afferents+by+spontaneous+activity+of+dorsal+horn+spinal+neurones+in+the+cat.">Modulation of synaptic transmission from segmental afferents by spontaneous activity of dorsal horn spinal neurones in the cat.</a> J. Physiol. 529 Pt 2, 445-460 (2000).</li><li>Geis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Stiff-Person+Syndrome+IgG+Induces+Anxious+Behavior+in+Rats.">Human Stiff-Person Syndrome IgG Induces Anxious Behavior in Rats.</a> PLoS One. 6, e16775 (2011).</li><li>Martinez-Gomez, J., Lopez-Garcia, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+pharmacological+characterisation+of+ascending+anterolateral+axons+in+the+in+vitro+mouse+spinal+cord.">Electrophysiological and pharmacological characterisation of ascending anterolateral axons in the in vitro mouse spinal cord.</a> J. Neurosci. Methods. 146, 84-90 (2005).</li></ol>

Extra- and intracellular electrophysiological recordings of neuronal activity and synaptic potentials in vivo are state of the art techniques in investigating CNS neuronal functions and pathophysiology. Spinal integration is critical for motor function, e.g. limb movement and for multimodal sensory perception. Presynaptic inhibition is one critical mechanism in this computational process ensuring appropriate responses to sensory inputs. GABAergic synapses on Ia afferent fibers inhibit the excitation of ...

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. It inhibits excitatory postsynaptic potentials (EPSPs) in monosynaptically excited motoneurons without changing the postsynaptic membrane potential and the excitability of the motoneurons1-3. Primary afferent depolarization (PAD) induced by GABAergic axo-axonal synapses onto sensory presynaptic fibers is the underlying mechanism4-7 (see also Figure1a). These synapses contain GABAA- and GABAB-receptors (GABAAR and GABABR). GABAAR activity leads to an increase in chloride conductance which elicits PAD due to the local ion distribution. This depolarization blocks the propagation of action potentials into the axon terminals and reduces their strength leading to a decreased Ca2+-influx and a reduction of transmitter release. Activation of GABAB receptors does not contribute to PAD but leads to a reduction of Ca2+-influx thereby enhancing presynaptic inhibition. While the activation of GABAAR seems to be involved in short term inhibition, GABABR are involved in long-term modulation8-10. In addition to GABA, which accounts for the major part of PAD and presynaptic inhibition, other transmitters systems might also modulate and contribute to this mechanism11,12.
Pathological changes in presynaptic inhibition seem to be crucial in several disease states e.g. peripheral inflammation and neuropathic pain13,14, as well as abnormal central pain processing15, spinal cord injury16, and CNS disease with motor hyperexcitability mediated by defective GABAergic transmission17,18. Thus, estimating presynaptic inhibition is worthwhile to investigate experimental pathological conditions on the spinal cord level in vivo. PAD gives rise to volume conducted potentials providing a direct measure of the presynaptic inhibition in the spinal cord. Those potentials are called dorsal root potentials (DRP) and can be measured from spinal cord dorsal roots after stimulation of adjacent dorsal roots7.
First measurements of DRP have been reported in cats and frogs19 and were intensively studied in cats by Eccles, Schmidt, and others in the early 1970s3,4,20,21. While in vivo recordings of DRP in cats22 and rats23 have been widely used, measurements in mice have been almost exclusively performed in ex vivo isolated spinal cord preparations15,24. Here, we describe a method to record DRP in anesthetized mice in vivo allowing a direct measure of presynaptic inhibition in the intact organism.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Typical DRP traces are shown in Figure 3. The prominent stimulation artifact is usually followed by a short downward deflection. Thereafter a slow, long-lasting upward deflection, representing the DRP is clearly distinguishable. In a subset of recordings, dorsal root reflexes are visible as small spikes on top of the DRP. In normal wild-type mice, dorsal root reflexes appear most often when stimulation voltage is excessive. As the dorsal root reflexes cannot be elicited with a high reproducibility in thi...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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